Ibrutinib treatment improves T cell number and function in CLL patients

Abstract

Background: Ibrutinib has been shown to have immunomodulatory effects by inhibiting Bruton's tyrosine kinase (BTK) and IL-2-inducible T cell kinase (ITK). The relative importance of inhibiting these 2 kinases has not been examined despite its relevance to immune-based therapies.

Methods: Peripheral blood mononuclear cells from chronic lymphocytic leukemia (CLL) patients on clinical trials of ibrutinib (BTK/ITK inhibitor; n = 19) or acalabrutinib (selective BTK inhibitor; n = 13) were collected serially. T cell phenotype, immune function, and CLL cell immunosuppressive capacity were evaluated.

Results: Ibrutinib markedly increased CD4+ and CD8+ T cell numbers in CLL patients. This effect was more prominent in effector/effector memory subsets and was not observed with acalabrutinib. Ex vivo studies demonstrated that this may be due to diminished activation-induced cell death through ITK inhibition. PD-1 and CTLA-4 expression was significantly markedly reduced in T cells by both agents. While the number of Treg cells remained unchanged, the ratio of these to conventional CD4+ T cells was reduced with ibrutinib, but not acalabrutinib. Both agents reduced expression of the immunosuppressive molecules CD200 and BTLA as well as IL-10 production by CLL cells.

Conclusions: Ibrutinib treatment increased the in vivo persistence of activated T cells, decreased the Treg/CD4+ T cell ratio, and diminished the immune-suppressive properties of CLL cells through BTK-dependent and -independent mechanisms. These features provide a strong rationale for combination immunotherapy approaches with ibrutinib in CLL and other cancers.

Trial registration: ClinicalTrials.gov NCT01589302 and NCT02029443. Samples described here were collected per OSU-0025.

Funding: The National Cancer Institute.

Conflict of interest statement

Conflict of interest: K.J. Maddocks received grants and nonfinancial support from Pharmacyclics, Merck, and Bristol-Myers Squibb (BMS), and personal fees from Pharmacyclics, BMS, and Janssen, outside the submitted work. J.A. Jones received personal fees and other support from Janssen, Pharmacyclics, and AbbVie, outside the submitted work. L.A. Andritsos received grants from Pharmacyclics during the conduct of the study. F. Awan received grants and personal fees from Pharmacyclics, personal fees from Gilead, and grants from Innate Pharma, outside the submitted work. J.A. Fraietta has a patent for the treatment of cancer using anti-CD19 chimeric antigen receptor licensed to the University of Pennsylvania. C.H. June received grants from Novartis during the conduct of the study and other support from Novartis and Tmunity Therapeutics, outside the submitted work. J.A. Woyach received research funding from Karyopharm Therapeutics, MorphoSys, AbbVie, and Acerta Pharma, outside the submitted work, and is on the advisory board for Janssen. A.J. Johnson has filed a patent on ibrutinib as immune-modulation agent. N. Muthusamy has filed a patent on ibrutinib as an immune-modulation agent. J.C. Byrd has filed a patent on ibrutinib as an immune-modulation agent.

Figures

Figure 1. Ibrutinib but not acalabrutinib treatment of CLL patients increases total T cell numbers.

(A) Absolute numbers of CD8+ (upper panel) and CD4+ (lower panel) T cells before and during ibrutinib treatment (n = 18). (B) Absolute numbers of CD8+ (upper panel) and CD4+ (lower panel) T cells before and during acalabrutinib treatment (n = 12). Each cycle is 4 weeks. Cycle 3 indicates samples obtained after 2 cycles (8 weeks into treatment), and cycle 6 indicates samples obtained after 5 cycles (20 weeks into treatment). T cells were differentiated into subsets based on expression of CCR7 and CD45RA: naive T cells (CCR7+CD45RA+), central memory T cells (CCR7+CD45RA–), effector memory T cells (CCR7–CD45RA–), and more differentiated effector memory T cells (T-EMRA; CCR7–CD45RA+). Differences were assessed using linear mixed-effects models. NS, not significant.

Figure 2. Ibrutinib treatment does not affect total numbers of stem memory T cells.

(A) Gating strategy for stem memory T cells. Naive CD4+ T cells (CCR7+CD45RA+) expressing CD62L were selected for further analysis. Of these, CD95+CD122hi cells were defined as stem memory T cells. (B) Left: Representative plots showing stem memory T cells in samples from a healthy donor and a CLL patient. Right: Representative plots showing T-bet and eomesodermin (eomes) expression in naive CD8+ and CD4+ T cells from a healthy donor and a CLL patient. (C) Stem memory T cells before and during ibrutinib treatment. Left: Graphs showing absolute numbers (top) and percentages (bottom) of stem memory cells (n = 15). Right: Representative plots. Differences were assessed using linear mixed-effects models. NS, not significant.

Figure 3. Treatment with BTK inhibitors significantly reduces PD-1–expressing cells in CD8+ T cell populations.

Percentages of PD-1+ cells among different subsets of CD8+ T cells from CLL patients before and during treatment with (A) ibrutinib (n = 17) or (B) acalabrutinib (n = 10). Differences were assessed using linear mixed-effects models. NS, not significant.

Figure 4. Treatment with BTK inhibitors significantly reduces the frequency of T cells expressing intracellular CTLA-4.

Percentages of cells positive for intracellular CTLA-4 expression among total CD4+ T cells, CD45RA– CD4+ T cells, and CD45RA+ CD4+ T cells from CLL patients before and during treatment with (A) ibrutinib (n = 18) and (B) acalabrutinib (n = 9). Differences were assessed using linear mixed-effects models. Data for CD8+ T cells are shown in Supplemental Figure 4.

Figure 5. Ibrutinib treatment does not affect T cell production of Th1 or Th2 cytokines but moderately increases frequency of Th17 cells.

PBMCs from CLL patients before and during treatment with (A) ibrutinib (n = 15) or (B) acalabrutinib (n = 11) were stimulated with PMA/ionomycin for 5 hours. Production of IFN-γ, TNF, IL-2, IL-4, and IL-17 was detected by intracellular cytokine staining. Percentages of cytokine-producing cells are shown. Differences were assessed using linear mixed-effects models. NS, not significant.

Figure 6. Ibrutinib treatment of CLL patients leads to a reduced frequency, but not reduced absolute number, of CD4+CD25+ Foxp3+ Treg cells.

(A) Representative plots showing CD25 and intranuclear Foxp3 staining in CD4+CD3+ (upper panel) and CD8+CD3+ (lower panel) T cells. (B and C) Percentages (left) and absolute numbers (right) of CD25+Foxp3+ regulatory T cells among total CD4+ T cells before and during treatment with (B) ibrutinib (n = 18) or (C) acalabrutinib (n = 11). Differences were assessed using linear mixed-effects models. NS, not significant.

Figure 7. Ibrutinib and acalabrutinib treatment of CLL patients reduces CD200 and BTLA expression in CLL cells.

Left panels: CD200 expression level as measured in mean fluorescence intensity (MFI) on CLL cells (CD19+CD5+) before and during treatment with (A) ibrutinib (n = 18) or (B) acalabrutinib (n = 12). Right panels: BTLA expression level as measured in MFI on CLL cells before and during treatment with (A) ibrutinib (n = 16) or (B) acalabrutinib (n = 12). Differences were assessed using linear mixed-effects models.

Figure 8. Ability of CLL cells to produce IL-10 is impaired with BTK inhibitor treatment.

PBMCs from CLL patients were collected before and during treatment with ibrutinib (A–C) and acalabrutinib (D). Cells were stimulated in vitro with CpG and PMA/ionomycin for 5 hours (B10 conditions) or with CpG/CD40L for 48 hours, with PMA/ionomycin added for the last 5 hours (B10Pro conditions). IL-10 production was detected by intracellular cytokine staining. All events were gated on CLL cells (CD19+CD5+CD3–). (A) Representative flow cytometry plots of IL-10 expression in CLL cells under B10 conditions (top) and B10Pro conditions (bottom). (B) CLL samples were collected at baseline and at the beginning of cycles 3 and 6 (8 and 20 weeks, respectively) after starting ibrutinib treatment. Graphs show the percentages of IL-10 producing CLL cells after in vitro incubation under B10 conditions (left; n = 13) and B10Pro conditions (right; n = 18). (C) PBMCs were collected from CLL patients at baseline and at the beginning of cycles 3 and 6 (8 and 20 weeks, respectively) after starting acalabrutinib treatment. Graphs show the percentages of IL-10–producing CLL cells after in vitro incubation under B10 conditions (left; n = 10) and B10Pro conditions (right; n = 12). Differences were assessed using linear mixed-effects models.